Mechanism Of Cadmium Induced Apoptosis Of Nucleus Pulposus-derived Mesenchymal Stem Cell In Intervertebral Disc Degeneration

Objective: With the rapid development of social aging,the incidence rate of low back pain in the population continues to rise,which has brought great economic and social burdens.Research has found that intervertebral disc degeneration(IVDD)is the main cause of low back pain,but the pathological mechanism of IVDD has not been fully clarified.In 2007,endogenous mesenchymal stem cells(MSC)in human intervertebral discs were first reported.Subsequently,nucleus pulposus-derived mesenchymal stem cells(NPMSC)were also found.Moreover,studies found that the number of endogenous NPMSC gradually decreased with the aggravation of IVDD,and the NPMSC isolated from degenerated intervertebral disc showed more apoptosis than those from normal intervertebral discs.Therefore,the apoptosis of NPMSC may be closely related to IVDD.Cadmium(Cd)is a toxic heavy metal and a global environmental and industrial pollutant.Cd exposure can cause damage to many organs and tissues of the human body,and bone tissue is one of the key target organs of Cd poisoning,which can lead to bone related diseases such as osteoarthritis and osteoporosis.The concentration of Cd in the blood of patients with spinal osteoarthritis was significantly higher than that of normal patients,indicating that Cd toxicity was closely related to the occurrence of spinal diseases.Moreover,Cd can induce apoptosis of annulus fibrosus cells of intervertebral disc,thus promoting IVDD.In conclusion,the cytotoxicity caused by Cd exposure may induce and aggravate IVDD.Therefore,this study aims to explore the effect of Cd on NPMSC and its possible mechanism,and provide a feasible target for preventing IVDD induced by Cd exposure.Experimental methods: Cell viability was detected by CCK-8,cell proliferation was detected by Ed U,cell survival rate was detected by Calcein/PI cell activity and cytotoxicity test kit,cell DNA damage was detected by TUNEL fluorescence staining,apoptosis ratio was detected by Annexin-V-FITC/PI apoptosis kit,differential genes and related pathways were detected by m RNA transcriptome sequencing,mitochondrial membrane potential was measured by JC-1 kit,and intracellular reactive oxygen species level was measured by DHE fluorescence probe,Adenosine triphosphate(ATP)detection kit was used to detect the intracellular ATP level,q PCR was used to detect the transcription of related genes,immunofluorescence analysis and Western blot analysis were used to detect the expression of related gene proteins.Results:1.Cd can inhibit the activity and proliferation of NPMSC in a dose-dependent and time-dependent manner,and promote cell aging and apoptosis.2.The mRNA transcriptome sequencing differential gene analysis,protein immunofluorescence and Western blot analysis showed that the gene expression of MAPK signaling pathway in Cd exposure group was significantly different compared with the control group,and the apoptosis rate of NPMSC was significantly decreased after the use of MAPK signaling pathway inhibitor BIRB 796.It suggests that Cd may promote the apoptosis of NPMSC by activating MAPK signaling pathway.3.Cd can increase the content of reactive oxygen species and the expression of cytochrome C protein in NPMSC,decrease the mitochondrial membrane potential and the level of intracellular adenine ATP.It suggests that Cd may mediate the apoptosis of NPMSC through mitochondrial pathway.Conclusion: Cd can inhibit the activity and proliferation of NPMSC,promote cell aging and apoptosis.At the same time,Cd may promote the apoptosis of NPMSC by activating MAPK signaling pathway.In addition,the destruction of mitochondrial homeostasis is also involved in the apoptosis of NPMSC.